RESUMO
Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells. Here, we induced MSCs from human induced pluripotent stem cells (iPSCs) via a neural crest cell (NCC) lineage under xeno-free conditions and evaluated their in vivo functions. We modified a previous MSC induction method to work under xeno-free conditions. Bovine serum albumin-containing NCC induction medium and fetal bovine serum-containing MSC induction medium were replaced with xeno-free medium. Through our optimized method, iPSCs differentiated into MSCs with high efficiency. To evaluate their in vivo activities, we transplanted the xeno-free-induced MSCs (XF-iMSCs) into mouse models for bone and skeletal muscle regeneration and confirmed their regenerative potency. These XF-iMSCs mainly promoted the regeneration of surrounding host cells, suggesting that they secrete soluble factors into affected regions. We also found that the peroxidasin and IGF2 secreted by the XF-iMSCs partially contributed to myotube differentiation. These results suggest that XF-iMSCs are important for future applications in regenerative medicine.
RESUMO
Somites (SMs) comprise a transient stem cell population that gives rise to multiple cell types, including dermatome (D), myotome (MYO), sclerotome (SCL) and syndetome (SYN) cells. Although several groups have reported induction protocols for MYO and SCL from pluripotent stem cells, no studies have demonstrated the induction of SYN and D from SMs. Here, we report systematic induction of these cells from human induced pluripotent stem cells (iPSCs) under chemically defined conditions. We also successfully induced cells with differentiation capacities similar to those of multipotent mesenchymal stromal cells (MSC-like cells) from SMs. To evaluate the usefulness of these protocols, we conducted disease modeling of fibrodysplasia ossificans progressiva (FOP), an inherited disease that is characterized by heterotopic endochondral ossification in soft tissues after birth. Importantly, FOP-iPSC-derived MSC-like cells showed enhanced chondrogenesis, whereas FOP-iPSC-derived SCL did not, possibly recapitulating normal embryonic skeletogenesis in FOP and cell-type specificity of FOP phenotypes. These results demonstrate the usefulness of multipotent SMs for disease modeling and future cell-based therapies.
Assuntos
Desenvolvimento Ósseo , Condrogênese , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Miosite Ossificante/metabolismo , Somitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Miosite Ossificante/patologia , Somitos/patologiaRESUMO
The neural crest is a source to produce multipotent neural crest stem cells that have a potential to differentiate into diverse cell types. The transcription factor SOX10 is expressed through early neural crest progenitors and stem cells in vertebrates. Here we report the generation of SOX10-Nano-lantern (NL) reporter human induced pluripotent stem cells (hiPS) by using CRISPR/Cas9 systems, that are beneficial to investigate the generation and maintenance of neural crest progenitor cells. SOX10-NL positive cells are produced transiently from hiPS cells by treatment with TGFß inhibitor SB431542 and GSK3 inhibitor CHIR99021. We found that all SOX10-NL-positive cells expressed an early neural crest marker NGFR, however SOX10-NL-positive cells purified from differentiated hiPS cells progressively attenuate their NL-expression under proliferation. We therefore attempted to maintain SOX10-NL-positive cells with additional signaling on the plane and sphere culture conditions. These SOX10-NL cells provide us to investigate mass culture with neural crest cells for stem cell research.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Crista Neural/metabolismo , Fatores de Transcrição SOXE/genética , Diferenciação Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Crista Neural/citologiaRESUMO
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-ß signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-ß signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.